This invention relates to a device for electrophoresis capable of analyzing extremely small quantities of samples at a very high speed and with a high resolution. More particularly, the invention relates to a device for electrophoresis referred to as a "microchip" having a separation flow route formed inside transparent planar members and throughholes provided to one of the surfaces at positions corresponding to both ends of the separation flow route and reaching to this flow route.
Devices for electrophoresis have been in use for analyzing a very small quantity of protein or nucleic acid, and those using a capillary have been representative examples. Devices of this type have a glass capillary with internal diameter less than 100 .mu.m and, after it is filled with a buffer and a sample is introduced at one of its ends, a high potential difference is applied between its ends and the target substance of analysis is dispersed inside the capillary. The application of a high potential is possible because the interior of the capillary has a relatively large surface area compared to its small volume and hence has a high cooling efficiency, and hence even a very small quantity of a sample such as DNA can be analyzed speedily and at a high resolution.
Since capillaries are easily breakable, having outer diameters as smaller as several 10-100 .mu.m, it is not an easy job for the user to exchange them. In view of this problem capillary devices for electrophoresis comprising two base plates joined together (referred to as the microchip) have been proposed as a form of device for electrophoresis which can take the place of the capillaries of the conventional kind, being capable of carrying out an analysis speedily and allowing the device to be easily miniaturized, as shown, for example, by D. J. Harrison et al (Anal, Chem. Acta 283 (1993) 361-366).
FIGS. 1A, 1B and 1C show an example of such a microchip 1, characterized as comprising a pair of transparent base plates 1a and 1b (for example, of glass, quartz or a resin material), mutually intersecting capillary grooves 3 and 5 being formed on a surface of one of these base plates (1b) and the other base plate (1a) being provided with reservoirs 7 in the form of a throughhole at positions corresponding to the end points of these grooves 3 and 5.
When the microchip 1 thus structured is used for carrying out an analysis, the two base plates 1a and 1b are stacked one on top of the other as shown in FIG. 1C, and a migration liquid is injected into the grooves 3 and 5 from one of the reservoirs 7. After a sample is injected into one of the reservoirs 7 at one of the ends of the shorter groove 3 serving as the "sample introducing flow route", a high potential difference is applied between the reservoirs at both ends of this groove 3 such that the sample is dispersed throughout the groove 3.
Thereafter, a migration potential difference is applied between the reservoirs 7 at both ends of the longer groove 5 serving as the "separation flow route" such that the portion of the sample at the intersecting area 9 of the two grooves 3 and 5 begins to migrate inside the longer groove 5. If an optical detector is disposed at a suitable position on the longer groove 5, the separated portions of the sample transported through electrophoresis can be sequentially detected thereby.
A problem with such a microchip is that the detection sensitivity is relatively low because the optical path length inside the separation flow route is short. In view of this problem, there has been proposed a new kind of microchip device for electrophoresis, as shown in FIG. 2, adapted to stop the application of the migration potential when target components in the sample to be detected have been separated or while they are being separated, to expose the entire separation flow route to a light beam, to use a linear image sensor or the like to repeatedly measure the light absorption of fluorescence and to carry out a multi-point averaging process on the measured values at different positions along the separation flow route such that the detection sensitivity can be improved.
Explained more in detail with reference to FIG. 2, a first mirror 13, a slit 15 and a second mirror 17 are disposed along the optical path of the light from a light source 11 for obtaining a parallel beam of light from the source 11. The parallel beam of light thus prepared is passed through a dispersion grating 19. There is provided a third mirror 21 serving to lead only a selected portion of the light dispersed by the grating 19 having a specified wavelength to the microchip 1. Disposed on the opposite side of the microchip 1 away from the light-incident side is a photodiode array (PDA) 23 with a plurality of linearly aligned photodiodes for measuring the light from the separation flow route. A high-voltage electric power source 25 is connected to the microchip 1. The PDA 23 is provided with a data processor 29 adapted to receive signals from the individual photodiodes of the PDA 23 through an analog-to-digital (A/D) converter 27 and to carry out multi-point averaging of signals for each photodiode.
When a sample is to be analyzed with such a device, the sample is injected at one end of the sample introducing flow route 3 of the microchip 1 and potential differences are applied from the power source 25 between the ends of the flow routes 3 and 5 as explained above to firstly bring the sample to the intersection area 9 of the flow routes 3 and 5 and then to complete the separation of target components to be analyzed inside the separation flow route 5. Thereafter, the application of the potential difference is stopped and a beam of monochromatic light is made incident within a specified region along the separation flow route 5. At each of specified positions along the separation flow route 5, the distribution of mutual interaction between the separated components and the monochromatic light such as absorption or fluorescence is repeatedly measured by the PDA 23, and the signals from the PDA 23 are analyzed by a multi-point averaging routine by the date processor 29 for each of the photodiode to achieve a detection with high sensitivity.
The microchip device for electrophoresis shown in FIG. 2 may be characterized as irradiating a specified area along the separation flow route 5 with a monochromatic beam of light with a wavelength selected by the grating 19. If a spectrum over a certain range of wavelengths is desired, however, it is necessary to vary a parameter (such as the angle of orientation) of the grating 19 and to thereby sequentially measure the reaction between the monochromatic light of different wavelengths and separated components. If it is desired to maintain the same level of detection sensitivity over the given range of wavelengths, measurement must be repeated at different wavelengths but the time required for the measurements becomes inconveniently long. Not only is it impossible to conclude the analysis quickly, but if the time for the measurements is prolonged under a separated condition, the level of separation is also adversely affected due to the natural diffusion of the separated components.